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Rescuing the Function of Mutant Von Hippel-Lindau Tumor Suppressor Protein using Stabilizing Small Molecules
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Thesis/Dissertation
Date
2025-05
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Biomedical Sciences
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DOI
https://doi.org/10.34944/tnbv-3076
Abstract
The Von Hippel-Lindau tumor suppressor gene, VHL, encodes for the pVHL tumor suppressor protein. While pVHL plays a role in many cellular activities, it is most well-known for its role in the oxygen sensing pathway. In this context, pVHL forms part of an E3 ubiquitin ligase complex that polyubiquitinates Hypoxia Inducible Factor α (HIF-α) for proteasomal degradation under normoxic conditions. Unsurprisingly, germline and somatic mutations in the VHL gene are strongly linked to several forms of cancer. These include the hereditary cancer syndrome, Von Hippel-Lindau Disease (VHLD), and sporadic clear cell renal cell carcinoma (ccRCC). I hypothesize that many clinically relevant missense mutations in the VHL gene thermodynamically destabilize the pVHL protein, resulting in a loss of protein foldedness and function. The central aim of this work is to identify small molecules that bind to and stabilize these pVHL mutants in the properly folded (native) conformation, thus restoring function.To that end, I utilized in silico methods to generate an ensemble of low energy, pocket containing conformations of pVHL (in the setting of the pVHL / Elongin C / Elongin B protein complex – hereby referred to as the “VCB” complex) and ranked these pockets in terms of druggability as predicted by a previously developed machine learning model. Notably, these computational studies identified the solvent filled pocket (and immediately adjacent areas) around Pro192, Gly127, and Asp197 as the most druggable site on the protein. The highly druggable pockets corresponding to this site were subsequently used as the basis for a virtual screen of an ultra-large virtual compound library to identify suitably complementary ligands capable of binding to these pockets. In the end, a series of 18 make on demand small molecules (known as “CP1-18”) were purchased for experimental validation.
The initial set of CP1-18 were screened for potential binding activity to purified VCB protein complex in pools of 4-5 compounds using Saturation Transfer Difference (STD) NMR techniques. One such hit, CP4, was selected for cellular testing by Dr. Mariam Fouad and demonstrated the ability to enhance thermal stability of wild type pVHL (via CETSA) and increase the cellular stability (via cycloheximide pulse chase assay) the missense mutant pVHL P86S. However, preliminary experiments failed to show robust ability to reduce HIF-2α protein levels in RCC-MF cells. To begin optimizing this CP4 for the ability to reduce HIF-2α, an initial round of SAR by catalog was conducted to identify a set of 32 CP4 derivatives from the Enamine REAL Space. Screening these compounds for the ability to decrease HIF-2α levels yielded CP4.29 that showed a marked improvement in terms of potency. Additionally, CP4.29 demonstrated the ability to reduce additional pVHL targets including ZHX2 and AURKA. Mechanistic studies involving co-treatments with a proteasome inhibitor, a NEDD8 activating enzyme inhibitor, and a prolyl hydroxylase inhibitor showed that CP4.29’s mechanism of action is consistent with direct pVHL reactivation. In hopes of further increasing potency, a series of rationally designed CP4.29 derivatives were synthesized but failed to demonstrate enhanced potency compared to CP4.29.
Having established CP4 and CP4.29 as the most promising compounds, rigorous confirmation of a direct interaction between CP4 and the VCB protein complex was conducted using a variety of orthogonal NMR based approaches. Having demonstrated a direct interaction between CP4 and VCB, attempts were made to determine the binding pose of CP4 using X-Ray crystallography. Unfortunately, these experiments were not successful. To determine the binding site of CP4, a point mutation was generated at the intended binding pocket (D197K) on pVHL and the same NMR experiments were repeated. These results showed a significant difference in binding kinetics between the wild type and pocket mutant constructs corroborating the computational models. These results, taken together, lay the foundation for a) pVHL reactivating small molecules as a novel potential therapeutic strategy for the treatment of ccRCC and VHLD and b) computational based screening approaches that may be generalized for the discovery of small molecules capable of reactivating other destabilized mutant proteins.
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